Biological pesticides are an alternative to those obtained synthetically, because they are not toxic to humans. Among them are those produced from entomopathogenic fungi, whose spores are dehydrated in order to remain viable for extended periods (Moore et al. Effects of moisture content and temperature on storage of Metarhizium flavoride conidia. Biocontrol Science and Technology, v. 6, p. 51-61). Dehydration also allows spores to survive in extreme environments characterized by dry heat, freezing and thawing, as well as acidic medium.
For more than a century research has shown the interaction between fungi and agricultural pests. Such interaction promotes the development of crop plants through the elimination of their pathogens, insect pests and weeds. Such findings have stimulated the use of mycopesticides to control agricultural pests. The production of biopesticides has increased and, among the causes of this increase are the demand of consumers for healthier foods, foods with less toxic waste, greater awareness of industry professionals regarding the use of agricultural pesticides, increasingly restrictive legislation on chemical pesticides and the need to use alternative products in programs to manage the resistance to chemical.
Fungi used in the biological control of pests and are used as pesticides are exposed to high temperatures, reaching 50° C. or more during transport or storage. This environmental factor affects the viability of fungal spores sensitive to temperature rises, such as Metarhizium, Beauveria, Lecanicillium and Trichoderma. The studies carried out to date were directed especially to storage of said fungi under refrigerated conditions or ambient temperatures below approximately 30° C. Mycopesticides experience rapid decline in viability during storage without refrigeration and it compromises product acceptance in the market, causing undesirable results in control of target pests.
The study by Marques and Alves (Marques, E J., Alves, S. R. “Otimizagao de formulações na preservagao de esporos de Beauveria bassiana (Bals.) Vuill. e Metarhizium anisopliae (Metschn.) Sorok em diferentes condições de armazenamento. [Optimization of formulations in the preservation of spores of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metschn.) Sorok at different storage conditions] Arquivos de Biologia e Tecnologia, v. 39, p. 861-877, 1996) demonstrated that the viability of spores having moisture content of 15.5% stored at 30° C. can be greatly reduced in less than 30 days.
The study of Sandhu et al. (Sandhu, S. S., Rajak, R. C., Agarwal, G. P. Studies on prolonged storage of Beauveria bassiana conidia: effects of temperature and relative humidity on conidial viability and virulence against chikpea borer. Helicoverpa armigera. Biocontrol Science and Technology, v. 3, p. 47-53, 1993) disclosed that the lower the relative temperature and humidity of the equilibrium adopted during storage, the longer the viability of Beauveria bassiaria spores is preserved.
Great emphasis has been given to the storage of entomopathogenic fungi and other species in environments with low or moderate temperature or in packages which allow exchange between the atmospheres inside and outside, which do not constitute suitable methods for storage at temperatures above 25° C.
Document U.S. Pat. No. 5,989,898 discloses the use of impermeable packages and humidity and oxygen absorbers to generate atmosphere with relative humidity of less than 10% and less than 5% oxygen. The document also proposes the elimination of oxygen by vacuum packaging, or by applying nitrogen to the package with spores.
The microorganisms used were Beauveria bassiana and Metarhizium anisopliae for storage at 25° C. and 37° C. Document U.S. Pat. No. 5,989,898 uses a surfactant agent to reactivate the spores, differing from the present invention, which allows storage at temperatures above 37° C., using different nontoxic gases (CO2, H2 and He) in substitution to oxygen, and adopts the observance of a pre-incubation period of the packaged product. It is important that properly packaged mycopesticides be exposed to suitable temperature conditions before being exposed to extreme conditions to allow the levels of oxygen and moisture to reduce to suitable levels. WO 9,718,294 discloses a two- to six-fold extension of the shelf-life of fungal spores or bacteria by reducing the oxygen content, associated or not with moisture reduction methods. However, the maximum storage temperature evaluated was 30° C., and after only 70 days of storage the initial viability was reduced by 85% or more in the treatment employing an O2 absorber sachet or in the treatment employing nitrogen. The present invention allows for maintenance of viability of fungal spores stored at higher temperatures, e.g. 40° C. over a period of three to six months.